Method using a new calibrator and a device and test kit including the calibrator

ABSTRACT

A lateral flow method for the determination of an analyte in a sample utilizes biospecific affinity reactions. The method comprises forming a complex comprising Reactant I - - - Analyte′ - - - Reactant* wherein Reactant* and Reactant I exhibit biospecific affinity to the analyte, Reactant* is analytically detectable, and Analyte′ is the analyte or an analyte-related reactant, determining a detectable signal from Reactant* in the complex (sample value), and obtaining the amount of analyte in a sample by comparing the sample value with one or more calibrator values, each of which corresponds to a standard amount of analyte. Before determination of the calibrator value, either calibrator or a binder for the calibrator has been bound to a matrix and is released at the determination of calibrator value. The calibrator and the analyte have the ability to biospecifically bind to Reactant* via equivalent binding sites, and one or more calibrator zones comprising calibrator or binder for the calibrator are located in the same process flow as Reactant I in a detection zone. A device for transforming measured signal values for a completed, analytically detectable reactant (Reactant*) to real amounts of analyte in a sample comprises a flow matrix in which there is an area of process flow for the transport of Reactant*, having one or more calibrator zones, an application zone for Reactant* upstream of the one or more calibrator zones, and one or more detection zones downstream of the one or more calibrator zones.

TECHNICAL FIELD

The invention relates to a method associated with a process for thedetermination of an analyte in a sample, which process involvesutilizing biospecific affinity reactions. The method includes the stepsof:

-   -   i. forming a complex containing:        -   Reactant I - - - Analyte′ - - - Reactant*, where            -   a. Reactant* and Reactant I exhibit biospecific affinity                to Analyte′, and            -   b. Reactant* is analytically detectable subsequently    -   ii. determining the detectable signal from Reactant* in the        complex (sample value), and    -   iii. obtaining the amount of analyte in the sample by comparing        the sample value with corresponding signal(s) (calibrator        value(s)) from Reactant*, which has separately been allowed to        bind to one or more amounts of a calibrator (calibrator        amounts), each one of which corresponding to a known amount of        analyte (standard amount(s)).

Analyte′ is the analyte as such (in the sample) or an analyte relatedreactant, i.e. an added biospecific affinity reactant, included in thecomplex in an amount which is related to the amount of analyte in thesample. Reactant* and Reactant I can bind Analyte′ at the same time.This means that they bind to spatially separated binding sites.

This type of analytical methods has been carried out in so-called flowmatrices, whereby reactants including analyte are transported in aprocess flow through the matrix (=flow methodology) to a detection zone(DZ) where Reactant* is captured in an amount related to the amount ofanalyte in the sample. Capture occurs via a reactant (Capturer) which isfirmly anchored to the matrix in DZ. That is, Capturer is bound viabonds which are stable under the conditions used to capture Reactant* inthe detection zone. The Capturer may be Reactant I or a reactant whichhas biospecific affinity to Reactant I or to another reactant, which inturn, optionally via one or more additional reactants, has biospecificaffinity to Reactant I.

By reactants (including analyte) exhibiting biospecific affinity(bioaffine reactants) is meant individual members of the reactant pairs:antigen/hapten-antibody; biotin-avidin/streptavidin; two complementarysingle chains of nucleic acid etc. As antibodies, antigen bindingantibody fragments such as Fab, F(ab)₂′, single chain Fv antibodies(scFv) etc. are considered. The reactants in question need not benaturally occurring but can also be synthetically preparedmolecules/binders.

The type of test methods in question has previously been used primarilyfor biospecific affinity reactants where at least one part of anemployed reactant pair has exhibited protein structure, in particular inconnection with so-called immunochemical determination procedures.

The biospecific affinity reactions are primarily performed in aqueousmedia (such as water).

Previously used calibrators

Conventionally, the calibrator and analyte have often both been able tobind to Reactant*. The binding sites in question on the calibrator forbinding to Reactant* often having binding properties equivalent tocorresponding binding sites on the analyte. In practice this means thatthe calibrator and the analyte have had binding sites which arestructurally equal or similar, and cross-react with each other withrespect to Reactant*. Binding sites which cross-react with each otherfor/about a given reactant are equivalent.

Calibrator amount has in the prior art commonly been equated withstandard amount.

Calibrator values, corresponding to different analyteamounts/concentrations (standard amounts), have often been compiled to adose-response curve (calibration curve) or an algorithm.

The expression “to compare a sample value with calibrator value(s)” hasalso encompassed that the comparison may take place with a calibrationcurve and/or algorithm, corresponding to several calibrator values.

The calibrator and the analyte have often been the same substance. Thereare exceptions. In antibody determination one and the same calibratorhas often been operational for several antibody specificities, providedthat the calibrator substance has been selected such that it exhibits aconstant domain of the antibody to be determined. See for example AbbottWO 97/27486.

Disadvantages of the prior art

The prior art has usually involved determination of several calibratorvalues in parallel with samples by running known amounts of analyte(standard amounts) in a way corresponding to samples. This has in turnled to 5-20% of all runs having been calibrator runs. By reducing thenumber of calibrator runs, possibly also by reducing the number ofreaction steps in each calibrator run, time and consumption of reagentcould be saved.

Often problems occur depending on calibrator and sample solutions havingdifferent properties and contents. This is particularly pronounced inimmunological tests where the calibrator often is measured in a buffer,and the analysis of sample is performed on serum or plasma samples. Adifference in contents and viscosity yields different responses (i.a.measured as “recovery” and parallelity). In addition the viscosity in aflow method becomes extra important since it influences themigration/flow velocity. This difference can be compensated for but atthe same time it renders the systems more sensitive to disturbances, andthus increased inter assay variation. Other problems with testsutilizing flows are possible flow variations depending on temperatureand moisture fluctuations etc.

The above problems have to some extent been overcome by the assay methoddisclosed in EP-A-253,464 and which uses a test zone and a referencezone on a solid phase.

OBJECT OF THE INVENTION

A first object of the invention is to improve the calibration methodspresently used in tests of the kind initially mentioned.

Another object of the invention is to simplify the use of calibrators,primarily by reducing the necessary consumption of reagents neededand/or reducing the number of measurements for obtaining calibratorvalues.

A third object of the invention, in particular in connection with flowmethods, is to enable compensation for the differences that may existbetween calibrator and sample solution and between runs performed atdifferent times and/or at different places.

THE INVENTION

We have now realized that these objects can be achieved if thecalibrator is bound to the matrix before beginning the determination ofcalibrator value in accordance with a relevant protocol. This type ofcalibrator will be referred to below as a matrix calibrator. The firstmain aspect of the invention is therefore a method in accordance withthe procedure mentioned initially, and which has the characterizingfeature that the calibrator, or a reactant capable of binding to thecalibrator, has been bound to a matrix which is insoluble in the liquidmedium in which binding of Reactant* to the calibrator occurs, beforebeginning the determination of a calibrator value. This means that thecalibrator or the calibrator binder, respectively, usually has beenbound to the matrix already by the manufacturer, such that the matrixcalibrator is delivered as a ready component in a kit. The bindingbetween calibrator and matrix normally is of another kind than thatobtained between Analyte′ and Reactant I when running a sample.

Matrix calibrators provide great advantages, if transport of Reactant*to the calibrator occurs by means of a flow (process flow) in aso-called flow matrix to a zone in the matrix, which contains the matrixcalibrator or the calibrator binder (calibrator zone, CZ).

When a calibrator binder is bound to the matrix, the calibrator may beeither movably (diffusively) pre-deposited in the matrix in a zoneseparated from the detection zone, or it may be added together with orseparately from the sample.

The calibrator binder is usually one member of a specific binding pair(reactant pair), the other member of the binding pair being coupled orconjugated to the calibrator substance. Such specific binding pairs arewell-known to a person skilled in the art, and as examples may bementioned: immunological binding pairs, such as antigen-antibody andhapten antibody, biotin-avidin or -streptavidin, lectin-sugar,hormone-hormone receptor, nucleic acid duplex.

Flow matrices

The flow matrix defines the space in which the reactants aretransported. Thus, the matrix may be the inner surface of a single flowchannel (such as a capillary), the inner surface of a porous matrixhaving a system of flow channels (porous matrix) etc. extending through.This type of matrices is called flow matrices. The matrices may exist inthe form of monoliths, sheets, columns, membranes, single flow channelshaving capillary dimensions, or aggregated systems of such flow channelsetc. They may also exist in the form of particles packed in columncasings, compressed fibers etc. The inner surface of the matrix, i.e.the surface of the flow channels, should be hydrophilic, such thataqueous media (primarily water) may be absorbed and transported throughthe matrix. The minimum inner dimension of the flow channels (measuredas a diameter for channels having a circular cross section) should besufficiently large for allowing transport through the matrix of thereactants being used. The rule of thumb is that suitable matrices areselectable among those having flow channels with the smallest innerdimension in the interval 0.4-1000 μm, preferably 0.4-100 μm if thematrix has a system of mutually communicating flow channels. Flowchannels having a smallest inner dimension in the upper part of thebroad interval (up to 1000 μm) are primarily of interest for flowsdriven by an externally imposed pressure/suction.

Matrices of interest are often built up from a polymer, e.g.nitrocellulose, nylon etc. The material in the matrix as well as thephysical and geometrical design of the flow channels may vary along theflow, depending on what a certain part of the matrix is to be used for(Pharmacia AB WO 96/22532; Medix WO 94/15215).

Along the flow in the matrix there may be one or more defined zones forapplication of sample, reactants, buffer etc. (A_(S)Z, A_(R)Z, A_(B)zetc.), and one or more zones for calibrator and/or detection (CZ and DZ,respectively).

Various flow matrices that may be used in the type of tests in questionare described in previous patent publications. See e.g. BehringwerkeU.S. Pat. No. 4,861,711, Unilever 88/08534, Abbott U.S. Pat. Nos.5,120,643 and U.S. Pat. No. 4,740,468, Becton Dickinson EP 284,232 andU.S. Pat. No. 4,855,240; Pharmacia AB WO 96/22532.

Process flow

The direction of the flow is from a zone of application of sample and/orreactant and towards existing calibrator and detection zones (CZ and DZ,respectively). Precisely which zones the process flow is to pass isdetermined by the test protocol in question. A process flow may startfrom a point with a radial spread and a flow front in the form of acircular periphery or a part thereof. A process flow may also start froma zone in the form of a band and may have a straight flow frontperpendicular to the direction of flow.

In a less preferred variant, the process flow proceeds from anapplication zone for Reactant*, which at the same time is a calibratorzone or a detection zone. In this variant the flow is preferablyradially spread from the zone of application, and may pass additionalcalibrator zones and/or detection zones.

Flow through the matrices may be achieved by influence from capillaryforces, e.g. by starting off with a substantially dry matrix. A suckingbody may be placed at the very end of the flow as an aid. By means of animposed electrical field, dissolved components may be transported fromthe zone of application to a detection/calibrator zone.

The utilized flow is preferably lateral, i.e. parallel with the uppersurface of the matrix. Also other types of flows, such as in depth inthe matrix, may be used.

Calibrator and detection zones in flow matrices

The flow matrix used in the preferred embodiment exhibits one or moredistinct zones with calibrator (calibrator zones, CZ1, CZ2, CZ3 etc.).Each calibrator zone contains matrix calibrator in an amount such thatthe measurement signal from Reactant* (calibrator value), detected inthe zone when a flow passes, distinctly corresponds to a certain amountof analyte in the sample (standard amount).

The calibrator may be selected in the same way as previously was thecase for the types of tests in question. Using flow methodology andarranging for sample (the analyte) to be transported through acalibrator zone, the calibrator should be selected such that it does notbind to the analyte. If the calibrator is able to bind analyte itimposes special requirements on the position of the calibrator zone inrelation to the zone of application of sample. See below.

The amount of calibrator that has bound to a calibrator zone does notneed to be the same as the corresponding standard amount. This isbecause the binding activity in relation to Reactant* often is changed,when the sample substance is bound to a matrix.

If it is desirable to determine antibodies with different specificitybut from the same species, of the same Ig class or Ig subclass, it ispreferred that the calibrator exhibits a binding site which is uniquefor the species, the class, or subclass. As a rule this means that acalibrator for determination of antibodies exhibits an epitope which ispresent in a constant domain of the antibodies in question, for mammalantibodies primarily a part of Ig(Fc).

One and the same matrix may exhibit one or more detection zones (DZ1,DZ2, DZ3 etc.) together with one or more calibrator zones. In thedetection zone, complexes containing Analyte′ and Reactant* bind to thematrix via the initially mentioned Capturer, which is firmly fixed in aDZ. If Reactant I binds to the matrix via the Capturer, Reactant I neednot be immobilized in the matrix from the start but may either bemovably (diffusively) pre-deposited in the matrix in an area or zoneseparated from the detection zone, or it may be added together with orseparately from the sample.

If there are several calibrator and/or detection zones in the same flowmatrix, the greatest advantages with the invention are achieved ifseveral of the zones are located along the same process flow.

If there are several detection zones (DZ1, DZ2, DZ3 etc.) in one and thesame matrix, these may correspond to different analytes. One can utilizethe same calibrator for analytes having equivalent binding sites. If theanalytes lack equivalent binding sites one calibrator is required foreach analyte. If all analytes have the same equivalent binding site thesimpliest condition will be at hand. The same calibrator, the samecalibrator zones and the same Reactant* may then be utilized for allanalytes.

Calibrator zones and detection zones may be geometrically designed invarious manners (rectangular, circular, linear, dot-shaped etc.). Thezones may have different configurations relative to each other. Goodconfigurations are such wherein a common flow consecutively orsimultaneously penetrates several zones, in particular zones ofdifferent kinds (DZ and CZ). An example of consecutive penetration isparallel zones located after each other in the same process flow. Anexample of simultaneous penetration is zones located next to each otheron the same circular periphery, where the process flow is radiallyspread from the centre of the corresponding circle. Combinations ofthese variants may be used, i.e. apart from zones on a circularperiphery there are also zones on the periphery of circles which areconcentric with the first-mentioned circular periphery. Simultaneouspenetration may also be achieved with a straight flow front havingdetection and calibrator zones located next to each other at the samedistance from the starting point of the process flow.

If several detection zones and/or calibrator zones are located in thesame process flow, a measurement signal for these zones may be obtainedin one and the same test run/reagent application. If there are severalcalibrator zones in the same process flow, a dose-response curve(calibration curve) or algorithm may be set up for the values obtainedfor the same application of Reactant*. A calibrator zone that existstogether with a detection zone in the same flow may function as apositive internal calibrator (PIC).

In one variant a matrix is utilized exhibiting at least one calibratorzone (CZ1, CZ2, etc. (positive internal calibrators)) and at least onedetection zone (DZ1, DZ2, etc.) in combination with one or moreseparately obtained calibrator values. The separately obtainedcalibrator values need not refer to the same conditions under which thesample is to be run. To the extent separate matrix values, calibrationcurve and algorithm are intended to be used during a longer period oftime, reference is made to master values, master curve and masteralgorithm, respectively.

The use of separately obtained calibrator values involves:

-   -   i. letting sample and Reactant* pass a detection zone (DZ) and a        positive internal calibrator (PIC, CZ) in a matrix exhibiting        both DZ and CZ,    -   ii. determining the measurement signal from a CZ (PIC value, CZ)        and from DZ,    -   iii. comparing the PIC value with corresponding separately        obtained calibrator value(s), whereby any deviations are a        measure of deviations between the conditions under which the        sample has been run, and the standard conditions applying to the        separate calibrator value(s),    -   iv. adapting the measured signal for the sample (sample value)        to the conditions applicable for the separately obtained        calibrator values, and then    -   v. obtaining the amount of analyte in the sample by comparing        the adapted measurement signal for the sample with the separate        calibrator value(s).

Alternatively, one may adapt the separate calibrator values todeviations in conditions and then directly compare a measured samplevalue with adapted calibrator values. This is equivalent to the steps(iv) and (v) above (called vice versa in claims). In the steps (iv) and(v) it is, of course, included as an alternative to adapt thecorresponding calibration curve or algorithm in order to calculate thelevel of analyte by comparing the sample value with either of these.

What has been said above, applies, of course, also to the case that abinder for the calibrator has been bound to the calibration zone(s) ofthe matrix.

A calibrator and detection zone in the same process flow will reduceprevious sources of error having been caused by differences in sampleand calibrator. A positive internal calibrator and several calibratorzones in the same process flow will completely or partly compensate forvariations in flows between separate runs. The spread in the measurementresult should be lower while internal as well as external factors may becompensated for completely or partly. The problem with sample andcalibrator having different compositions is eliminated. For near patienttests, the internal calibrator will be able to provide a well definedlimit as to what constitutes a positive response, and to provide thequality assurance which today is missing for these types of tests.

The anchoring of the calibrator to the matrix may take place viacovalent bonding or via physical adsorption, biospecific affinity etc.Like prior art in this field the invention may utilize combinations ofbinding types, such as covalent bonding to the matrix of a biospecificaffinity reactant directed to the calibrator. Specifically, physicallyadsorbed or covalently bonded streptavidin in combination with abiotinylated calibrator may be mentioned, or a similarly bound antibodydirected to the calibrator. Anchoring of the calibrator to the matrixmay take place via particles having been deposited in/on the matrix, andto which the calibrator is covalently, physically adsorptively orbiospecifically etc. bound. The particles attach to the matrix eitherbecause their size has been selected such that they cannot betransported through the matrix, or via physical adsorption. See i.a.Abbott/Syntex U.S. Pat. No. 4,740,468; Abbott EP 472,376; Hybritech EP437,287 and EP 200,381; Grace & Co EP 420,053; Fuji Photo Film U.S. Pat.No. 4,657,739; Boehringer Mannheim WO 94/06012.

The Capturer may be bound to a detection zone according to the sameprinciples as those applying to a calibrator. In one and the sameprocess flow a calibrator and Capturer may be bound to their respectivezones in the same way or in different ways. What has been said aboveconcerning the anchoring of the calibrator and the Capturer, is, ofcourse, also applicable to the anchoring of a binder for the calibratorsubstance. For example, the above mentioned combination of abiotinylated calibrator substance and physically or covalently boundstreptavidin may be used.

Zone of application of sample (A_(S)Z)

The zone of application of sample may be located upstream or downstreamin relation to calibrator zones, preferably upstream. In case the matrixcalibrator has been selected such that it binds analyte, the zone ofapplication of sample must be located downstream of the matrixcalibrator. In relation to detection zones the zone of application ofsample should always be located upstream in useful embodiments.

In certain less preferred embodiments it is conceivable to apply samplein a calibrator or detection zone.

Zone of application of Reactant* (A_(R*)Z) and other biospecificaffinity reactants (A_(R)Z)

An application zone for Reactant* (A_(R*)Z) should always be locatedupstream of the calibrator zones.

If there is a detection zone in the process flow, the order of the zonesof application of biospecific affinity reactants should ensure thatAnalyte′ is transported into its detection zone before or simultaneouslywith Reactant*. One or more reactants may be added in the same zone ofapplication. If the zone of application is common to sample and at leastone reactant, let us say Reactant*, application may occursimultaneously, e.g. by having mixed a sample and a reactant before theyare applied in the zone. If desired, the mixture may be preincubatedsuch that the reactant will bind to the analyte or to other componentsin the sample, as intended, before application of the sample. Havingknowledge of various protocols, the skilled person will be able toeasily determine which zones he needs and the possible order thereof.

If Reactant I is present in dissolved form, the matrix has a zone ofapplication for it at the same time as there is a Capturer firmly fixedin the detection zone. If the Capturer requires additional biospecificaffinity reactants in order to bind Reactant I (see under “Technicalfield”), there are zones of application for these reactants. Zones ofapplication for Reactant I, when it is not a Capturer, and anyadditional reactants must be positioned such that Reactant I reaches thedetection zone before or at the same time as Analyte′. If Reactant I isin soluble form, the Capturer may preferably be one member of a specificbinding pair, the other member of which is coupled or conjugated toReactant I. Exemplary specific binding pairs are immunological bindingpairs, such as antigen-antibody and hapten-antibody, biotin-avidin or-streptavidin, lectin-sugar, hormone-hormone receptor, nucleic acidduplex.

If both the calibrator and Reactant I are in soluble form to then bindto the matrix via specific binding pairs, these two binding pairs are,of course, different.

In certain less preferred embodiments biospecific affinity reactants(inclusive Reactant*) may be applied in a calibrator or detection zone.See under the heading “Process flow”.

Reactants utilized in the method may be predeposited in their respectivezone or may be added in connection with performing the method ofdetermination. Predepositing involves application of the reactant inquestion in advance in such a way that it will not spread outside itszone of application until a flow of liquid is initiated in or passes thezone.

Predeposition of reactants may take place by methods known per se. Seefor example (Behringwerke U.S. Pat. No. 4,861,711; Unilever WO 88/08534;Abbott U.S. Pat. No. 5,120,643; Becton Dickinson EP 284,232). It isimportant to take into consideration the fact that a predepositedreactant should easily dissolve when liquid passes through the zone ofapplication in question. In order to achieve quick dissolution it iscommon to incorporate/codeposit reactants in/with substances thatquickly dissolve. This type of substances are often hydrophilic havingpolar and/or charged groups, such as hydroxy, carboxy, amino, sulphonateetc. In particular there may be mentioned hydrophilic quickly solublepolymers, e.g. having carbohydrate structure, simple sugars includingmono-, di- and oligosaccharides and corresponding sugar alcohols(mannitol, sorbitol etc.). It is common practice to first coat the zoneof application in question with a layer of the quickly solublesubstance, whereupon the reactant is applied, possibly followed by oneadditional layer of quickly soluble substance. An alternative way is byincorporating the reactant in particles of quickly soluble material,which then is deposited in the zone in question of the matrix.

Zones for buffer (A_(B)Z)

Buffer systems that are required may be included in solutions addedsimultaneously with samples and reactants. In conventional techniquesaddition of buffer takes place in the zone of application that islocated upstream of all other zones of application. This has often beenequal to the sample application zone. In the present invention buffermay in principle be added in an optional position along the flow oftransport. See below.

In a co-pending PCT application “Analytical method comprising additionin two or more positions and a device and test kit therefor” (based onSE 9704934-0) there is disclosed an invention, which in one variantprovides a preferred embodiment of the present invention. Thisapplication is hereby incorporated by reference in the present text. Theinvention in this separate patent application is based on the discoverythat liquid from two subsequent zones (AZ2 and AZ1) in a flow matrix maymigrate after each other without mixing. This will be achieved if liquidis applied to the zone (AZ1) located downstream before or essentiallysimultaneously with application of liquid to the zone (AZ2) locatedupstream. This discovery has led to the ability to achieve zonewisemigration of any reactants present in the liquids, towards a detectionzone. If the zone of application of sample (A_(S)Z) is locateddownstream of the zone of application of Reactant* (A_(R*)Z), and ifliquid is applied to A_(R*)Z and sample to A_(S)Z, the analyte maymigrate into the detection zone before the liquid containing Reactant*does. If there is one zone of application for liquid alone (buffer)(A_(B)Z) between A_(R*)Z and A_(S)Z, a wash of the detection zone DZ isobtained between capture of analyte and Reactant*. Such an intermediatebuffer zone (A_(B)Z) may also ensure that a reactant (includinganalyte), that is applied in a zone located downstream, reaches DZbefore a reactant, starting from a zone of application located upstream.The latter may be important if the matrix as such retards the reactantthat has been applied in the zone located downstream.

Reactants may be included in the liquid that is applied to a zone.Alternatively they may be pre-deposited in the zone where thecorresponding liquid is to be applied, or in a zone that is locatedbetween this and the nearest zone that is located downstream, forapplication of liquid. Sample (the analyte) normally is applied in theform of liquid.

This embodiment of the invention is particularly interesting forsequential methods of the type is question in flow matrices, i.e.methods wherein the matrix in addition to a calibrator zone alsocontains a detection zone, and where the sample/analyte is to betransported into the detection zone before liquid containing Reactant*.

Analytically detectable reactant (Reactant*)

Usually analytical detectability of a reactant is obtained because itcomprises an analytically detectable group. Well-known examples of oftenused groups are enzymatically active groups (enzyme, cofactor, coenzyme,enzyme substrate etc.), fluorophore, chromophore, chemiluminescent,radioactive groups etc. Groups being detected by means of a biospecificaffinity reactant are also usually referred to this category, e.g.biotin, hapten, Ig-class, Ig-subclass and Ig-species specificdeterminants etc. In this invention, particles the surfaces of whichhave been coated with a biospecific affinity reactant have proved to beparticularly good. The particles may contain any of the previouslymentioned detectable groups, such as fluorophore groups, or they may becoloured (=containing chromogenic groups). Useful particles often have asize in the interval 0.001-5 μm, preferably 0.01-5 μm. The particles maybe spherical and/or monodisperse or polydisperse. They may havecolloidal dimensions, so-called sol (i.e. usually spherical andmonodisperse having a size in the interval 0.001-1 μm). Well-knownparticulate label groups are metal particles (such as gold sol),non-metal particles (such as SiO₂, carbon, latex and killed erythrocytesand bacteria). In certain cases it has been emphasized that theparticles should be non-sedimentable under the utilized conditions. (SeePharmacia AB, WO 96/22532).

See also Unilever, WO 88/08534; Abbott, U.S. Pat. No. 5,120,643; BectonDickinson, EP 284,232.

In connection with the development of matrix calibrators we havesurprisingly found that good results may be obtained if onesimultaneously utilizes:

-   -   (a) Reactant* where the detectable group is particles as        disclosed above, and    -   (b) a detection zone in which the Capturer attaches to the        matrix via particles (anchoring particles), having dimensions        that would allow transport of the particles through the matrix.

We have achieved a functioning system wherein label particles andanchoring particles have had substantially the same dimensions, whichmeans that in all probability the label particles may be larger than theanchoring particles and vice versa, as long as they remain smaller thanthe flow channels defined by the matrix. The system may function with aswell as without predeposition of Reactant*. This embodiment is describedin more detail in a co-pending PCT-application “Analytical method usingparticles and test kit for performing the method” (based on SE9704935-7). Also this latter application is incorporated by reference.Applied to the present invention this means that Reactant* has particlesas an analytically detectable group according to a above, and that thecalibrator and/or the Capturer binds to the matrix via particlesaccording to b above.

Relevant test protocols

The invention may primarily be applied to non-competitive(non-inhibition) test variants, but also to competitive (inhibition)test variants, if these involve that a complex is formed with ananalyte-related reactant bound between Reactant I and Reactant*. Theprotocols may be run as simultaneous or sequential variants. Bysimultaneous methods is meant that Reactant* and Analyte′ areco-transported during at least a part of the transport towards thedetection zone, and preferably reach the latter simultaneously. Bysequential method is means that Analyte′ during at least a part of thetransport towards the detection zone migrates in front of Reactant*, andpreferably reaches the detection zone before Reactant*. Illustrativeexamples are given below. “-” relates to firm anchoring to the matrixfrom the start. “ - - - ” relates to binding via biospecific affinity.It has been assumed that the reactants are monofunctional with regard tothe binding sites being utilized.

A. Sandwich protocol: Reactant I (=Capturer) and Reactant* havebiospecific affinity to the analyte (=Analyte′). x is the number ofmoles of Reactant I on the matrix. y is the number of moles of Analyte′(=moles of Reactant*) that has been captured on the matrix via ReactantI.

-   -   Formed complex:        Matrix[−Reactant I]_(x-y)[−Reactant I - - - Analyte′ - - -        Reactant*]_(y)

B. Sandwich protocol: Reactant II (=Capturer) has biospecific affinityto Reactant I, which in turn has biospecific affinity to the analyte(=Analyte′). Reactant* has biospecific affinity to the analyte. x is thenumber of moles of Reactant II on the matrix. y is the number of molesof Analyte′ (=moles of Reactant*) that has been captured on the matrixvia Reactant II - - - Reactant I. z+y is the number of moles of ReactantI that has been captured on the matrix via Reactant II.

-   -   Formed complex:        Matrix[−Reactant II]_(x-z-y)[−Reactant II - - - Reactant        I]_(z)-[−Reactant II - - - Reactant I - - - Analyte′ - - -        Reactant*]_(y)

C. Protocol of inhibition type: Reactant I is an analyte analogue(=Capturer) and has binding sites that are equivalent with the bindingsites on the analyte. Analyte′ is a reactant that has biospecificaffinity to the analyte and to Reactant I. Reactant* has biospecificaffinity to Analyte′. Analyte′ is included in the formed complex in anamount that is related to the amount of analyte in the sample. x is thenumber of moles of Reactant I on the matrix. y is the number of moles ofAnalyte′ (=number of moles of Reactant*) that has been captured on thematrix via Reactant I.

-   -   Formed complex:        Matrix[−Reactant I]_(x-y)[−Reactant I - - - Analyte′ - - -        Reactant*]_(y)        Analytes in sample

The invention is primarily adapted for determination of bio-specificaffinity reactants (analytes) of the types mentioned initially. Greatadvantages are obtained for analytes occurring in multiple forms, whichhave as a common denominator at least one binding site with equivalentbinding properties.

For non-competitive methods (sandwich) the analyte may be an antibodydirected to an antigen (including allergen), or hapten (Test protocols Aand B above). Reactant I in this case is the antigen or the hapten towhich the antibody is directed, and Reactant* is an antibody directed tothe analyte. Alternatively Reactant* is the antigen or the hapten, andReactant I is an antibody directed to the analyte. For non-competitivemethods the analyte may also be an antigen, Reactant* and Reactant Ibeing antibodies directed to the antigen. As examples of analyte-antigenmay be mentioned immunoglobulin, possibly of a particular Ig class or Igsubclass. When the analyte is an antibody or an immunoglobulin,Reactant* and Reactant I, respectively, may exhibit biospecific affinitytowards an Ig determinant that is specific for an Ig class such as IgA,IgD, IgE, IgG or IgM and/or for a subclass if present (e.g. IgG1, IgG2,IgG3 or IgG4), and/or for a certain species. This means that Reactant*and Reactant I, respectively, normally is an antibody exhibiting some ofthese specificities when the analyte is an antibody or animmunoglobulin.

Competitive variants are primarily applicable to low molecular analytes.In the test protocol C above the analyte may be an antigen/hapten, inwhich case Reactant I is the antigen/hapten bound to the matrix,Analyte′ is an antibody directed to the antigen/hapten, and Reactant* isan antibody directed to Analyte′.

It has been particularly interesting for the inventors to be able tomeasure analytes the occurrence and/or amount of which being related toautoimmune diseases and allergy. It is particularly interesting tomeasure anti-allergen antibodies of IgE of IgG class, for the latterpreferably with emphasis on some of the mentioned subclasses.Measurement of allergen specific antibodies may be employed inconnection with diagnosing of IgE mediated allergy.

Samples

Relevant samples may be of biological origin, e.g. from different bodyfluids (whole blood, serum, plasma, saliva, urine, tear liquid,cerebrospinal fluid etc.), extracts from biological tissue, from cellculture media, processing procedures in biotechnology, from foodstuff,from the environment (environmental analysis samples) etc. The samplesmay be pretreated in order to fit e.g. the matrix, the test protocolinvolved etc.

A second aspect of the invention

This aspect of the invention relates to a test device where the matrixcalibrator constitutes a central point. The matrix calibrator is used inanalytical methods for transferring measured signal values (samplevalues) for a complexed, analytically detectable reactant (=Reactant*)to real amounts of analyte in a sample, in connection with performing ananalytical method utilizing biospecific affinity reactions. As in themethod aspect Reactant* is complexed in an amount that is related to theamount of analyte in a sample. The most important type of analyticalmethods for which the device may be used are those for which the methodof the invention is used, that is methods where one forms complexescomprising Reactant I - - - Analyte′ - - - Reactant*. Reactant I,Analyte′, Reactant* and - - - have the same meanings as in the methodaspect.

The device is characterized by exhibiting:

-   -   a) a flow matrix in which there is an area of process flow for        transport of Reactant*, and in that this area comprises        -   i. one or more calibrator zones (CZ1, CZ2 etc.) comprising a            calibrator, or a binder for the calibrator, that is firmly            anchored to the matrix, the amounts of calibrator or            calibrator binder, respectively, being different for at            least two calibrator zones, and the calibrator exhibiting            binding sites to which Reactant* may bind, when Reactant* is            transported through a calibrator zone, and        -   ii. an application zone for Reactant* (A_(R*)Z) located            upstream of said one or more calibrator zones.

If the calibrator zone/zones instead of the calibrator contains a binderfor calibrator substance, the device preferably also contains:

-   -   b) calibrator which is movably (diffusively) predeposited in or        downstream of A_(S)Z.        Preferably, the device is included in a kit which comprises:    -   c) Reactant* which may be predeposited in A_(R*)Z.

The process flow may also contain (a) a detection zone (DZ) locateddownstream or coinciding with A_(R*)Z, and in which there is a firmlyfixed Capturer via which Reactant* may bind to DZ, and (b) a zone ofapplication for sample (A_(S)Z) located upstream or coinciding with saidDZ. A_(R*)Z may be located upstream or downstream or coincide withA_(S)Z (if present), preferably upstream or downstream. If A_(S)Z and DZare present in the same process flow as the calibrator zone, A_(S)Z ispreferably located upstream and DZ preferably downstream of existingcalibrator zones.

In preferred embodiments the firmly anchored reactant (Capturer) hasbiospecific affinity to the analyte or to an analyte-related reactantthat may be analytically detectable. Analyte related reactant isprimarily relevant to competitive test variants.

Calibrator substance is selected in the same way as in the method aspectof the invention. In those cases where the selected calibrator substanceexhibits biospecific affinity to the analyte, the correspondingcalibrator zone shall be located upstream of A_(S)Z.

Additional details regarding calibrators, zones, reactants, matrices,process flows, test protocols, samples etc. are apparent from thedescription of the method aspect of the invention.

The invention will now be illustrated with a number of examples showingvarious preferred embodiments thereof. The invention is defined by theattached claims and what is disclosed in the description.

EXAMPLE 1 Determination of Birch Specific IgE with Carbon ParticleConjugate and with Calibrator Bound to the Matrix

Methods and materials

Adsorption of phenyldextran to polystyrene particles: Phenyldextran(degree of substitution: 1 phenyl group on each fifth monosaccharideunit=20%, Mw dextran 40,000, Pharmacia Biotech AB, Uppsala, Sweden) wasadsorbed to polystyrene particles (0.49 μm Bangs Laboratories, USA) byincubations under stirring with phenyldextran dissolved in deionizedwater to 1) 5 mg/ml, 10% particle suspension, RT 1 h, 2) 5 mg/ml, 5%particle suspension, RT 1 h, 3) 20 mg/ml, 1% particle suspension, RTovernight 15 h. The particles were subsequently washed twice withdeionized water. The particle suspensions were centrifuged between eachincubation and wash (12,100×g, 25 min, Beckman, J-21, JA-20, 10,000rpm). The particle suspension was finally sonicated (Ultrasonic bath,Branson 5210, 5 min).

Coupling of human IgE (hIgE) to polystyrene particle (=hIgE particles):Human IgE was coupled to phenyldextran coated polystyrene particles withCDAP (1-cyano-4-dimethylamino-pyridinium bromide (Kohn J and Wilchek M,FEBS Letters 154(1), (1983) 209-210).

Desalting and change of buffer of hIgE were performed by gel filtration(PD-10, Pharmacia Biotech AB, Sweden) in NaHCO₃, 0.1 M, pH 8.5. 278 mgof polystyrene particles (as above) in 2% solution in 30% (by volume)acetone were activated with 4.2 ml CDAP (0.44 M) and 3.4 ml TEA (0.2 Mtriethylamine, Riedel-de Haen, Germany). CDAP was added during stirringfor 60 s and TEA during 120 s. The particles were washed with 30% (byvolume) acetone and centrifuged at 12,100×g (25 min, Beckman, J-21,JA-20, 10,000 rpm). 25 mg of hIgE were coupled to the activatedparticles in incubation with stirring overnight at +4° C. Then theparticles were centrifuged before deactivating with glutamic acid 0.05 Mand aspartic acid 0.05 M in NaHCO₃ buffer. Incubation was performed withstirring overnight at +4° C. Coupled particles were washed with 0.1 MNaHCO₃ and twice with 20 mM borate buffer pH 8.5. The particleconcentration was determined spectrophotometrically at A₆₀₀ nm withuntreated particles as reference. Concentration of coupled protein wasdetermined by having radioactive human IgE present during coupling.

Extraction of t3 (birch pollen, Betula verrucosa): 1 part (weight) ofbirch pollen (Allergon, Sweden) was extracted with 10 parts (volume) 0.1M of phosphate buffer (denoted 1/10), pH 7.4. The extraction lasted for2 hours on a shaker table at +4° C. The extract was centrifuged at 4000rpm for 1.75 h. After filtering the solution was applied to a PD-10column and eluted in 0.1 M NaHCO₃, pH 8.5 (denoted 1/14).

Coupling of t3 extract to a polystyrene particle (t3 particles: t3extract (1/14) was coupled with CDAP (Kohn and Wilchek, FEBS Letters154(1) (1983) 209-210) to polystyrene particles coated withphenyldextran. Polystyrene particles (400 mg, coated with phenyldextranas above) in 30% (by volume) acetone, 2% particle suspension, wereactivated with 60 mg of CDAP (100 mg/ml in 30% acetone) and 0.48 ml 0.2M triethylamine (Riedel-de Haen, Germany). CDAP was added with stirringand TEA was added dropwise during 90 seconds and stirring for 120 s intotal. The reaction was quenched by the addition of 30% acetone (4 timesvolume) and centrifuging at 12,400×g for 35 min. The particles werewashed once with deionized water. 32 ml of t3 extract in 0.1 M NaHCO₃,pH 8.5, were added to 80 mg of the activated particles and coupling wascontinued for 1 hour on a shaker table. Then the particles werecentrifuged before they were deactivated with 0.05 M aspartic acid och0.05 M glutamic acid in 0.1 M NaHCO₃, pH 8.5. Incubation on shaker tableovernight at +4° C. The particles were washed by centrifuging in 1) 0.1M NaHCO₃, 0.3 M NaCl, pH 8.5; 2) 0.1 M Na acetate, 0.3 M NaCl, pH 5; 3)0.1 M NaHCO₃, pH 8.5; and 4) 20 mM Na borate, pH 8.5.

The particle concentration was determined spectrophotometrically at 600nm with uncoated polystyrene particles as reference.

Adsorption of anti-human IgE antibody to carbon particles (carbonparticle conjugate=Reactant*): Monoclonal anti-hIgE was adsorbed tocarbon particles (sp100 from Degussa, Germany). See Pharmacia AB, WO96/22532. The ready suspension was diluted with buffer to 400 μg ofcarbon particles per ml.

Deposition of t3 particles on membrane in a detection zone: One sheetson nitrocellulose with polyester backing (Whatman, 8 μm, 5 cm wide) 4%of the above-mentioned t3-coupled particles were applied with LinearStriper (IVEK Corporation) with a flow of 1 μl/s and 1 μl/cm as astraight zone. The sheets were dried for 1 hour, 30° C., whereupon thesheets were cut at right angles relative to the zone to 0.5 cm widestrips (Matrix 1201 Membrane Cutter, Kinematics Automation).

Deposition of hIgE particles in calibrator zones: On nitrocellulosesheets with polyester backing (Whatman, 8 μm, 5 cm wide) hIgE particleswere deposited as parallel calibrator zones with Linear Striper (IVEKCorporation, USA). The flow was 1 μl/s and 1 μl/cm. Sheets intended forstrips having only calibrator zones were coated with six parallel zones.hIgE concentrations in the zones were 0, 0.84; 3.4; 14; 54.2 and 217 nghIgE/0.5 cm. Before performing the deposition the hIgE particles werediluted in borate buffer (20 mM, pH 8.5, Dextran T5000 4.2% w/w,sorbitol 5.8% w/w). All zones also comprised 1% phenyldextran-coatedparticles in order to yield the same amount of particles in each zone.On a separate nitrocellulose sheet there was deposited a zone with hIgEparticles (14 ng hIgE/0.5 cm, PIC=positive internal calibrator), and ina parallel zone t3 particles as above (detection zone). The depositiontook place with the same parameter as for hIgE particles. The sheetswere dried 1 h, 30° C., and were then cut, perpendicularly relative tothe zones, to strips 0.5 cm wide (Singulator: Matrix 1201 membranecutter, Kinematic automation, USA).

Test procedure: Strips were mounted on a plane plastic surface. At thetop (0.5 cm) on the strip a sucking membrane was placed (width 3 cm,Whatman, 17 Chr). To obtain a constant pressure metal weights were puton the sucking membranes. 10 mm from the lower edge a 2 mm wideInplastor strip was mounted (preglued polyester film). The Inplastorstrip should prevent applied liquids from flowing out over too large aportion of the membrane. To the lower end of the strip there was applied30 μl of sample or buffer in the alternative. After suction of thesample volume the following components were applied in the given order:15 μl buffer, 15 μl carbon particle conjugate as above and 30+30 μlbuffer. The buffer was: NaPO₄ 0.1 M, BSA 3%, NaN₃ 0.05%, sucrose 3%,NaCl 0.5%, phenyldextran 0.25%, bovine gammaglobulin 0.05%, pH 7.5. Thedegree of blackening of the reaction zones was measured as absorbancewith ultroscan (Ultroscan XL, Enhanced Laser Densitometer, LKB).

Results

A) Activity determination on deposited IgE calibrator curve against IgEcalibrators (24° C.) run as samples on separate strips with anti-hIgEantibody in the binding zone.

TABLE 1 Deposited Calculated amount IgE KU/L Abs (x1000)* 1 0.84 0.27 46 2 3.4 0.48 109 3 14 0.71 266 used below as positive internalcalibrator 4 54.2 2.7 619 5 217 66.3 1882  *= absorbance on a reactionzone after carbon particle conjugate having bound.

B) Determination of birch specific IgE antibody in patient samples runat 18, 24 and 37° C., with and without positive internal calibrator(PIC) in order to adjust the standard curve (run at 24° C.).

TABLE 2 Results (KU/L) with and without corrected calibrator curve:Corrected Not corrected 18° C. 24° C. 37° C. 18° C. 24° C. 37° C. Sample1 1.3 1.1 1.4 0.83 1.1 1.8 Sample 2 6.9 5.5 6.7 5.1 8.6 20

The results show that it is possible to compensate for the variation inthe separate runs by using positive internal calibrators. In additionthe results show that it is possible to use predeposited calibrators.

The embodiment shown in this example may be modified such that one ormore of the following criteria are met, (a) has predeposited Reactant*in a zone of application and/or (b) has a zone of application of samplelocated downstream or upstream of the zone of application of Reactant*,(c) has zones allowing simultaneous addition of Reactant* and sample.

EXAMPLE 2 Determination of Birch-Specific IgE with Fluorescent DetectionParticles and with a Calibrator Predeposited in the Application Zone

Methods and materials

Coupling of streptavidin to polystyrene particles: Streptavidin(Amersham Pharmacia Biotech AB, Sweden) were covalently coupled tophenyldextran-adsorbed polystyrene particles with CDAP(1-cyano-4-dimethylaminopyridinium bromide) (Kohn J and Wilchek M, FEBSLetters 154 (1) (1983) 209-210), according to the description in Example1 above for hIgE. The coupled particles were washed three times with 50mM NaPO₄, 0.05% NaN₃, pH 7.4. The particle concentration was determinedspectrophotometrically at A600 nm with untreated particles as reference.

Deposition of streptavidin-coupled particles on nitrocellulosemembranes: To nitrocellulose sheets with polyester backing (Whatman, 8μm, 5 cm wide) were applied with Linear Striper (IVEK Corporation) zonesof:

-   -   1) streptavidin-coupled particles diluted to 1% particle content        i 10 mM NaPO₄, 5% sucrose, 5% dextran T5000, 0.01% NaN₃, pH 7.4;    -   2) t3-coupled particles, prepared according to Example 1,        diluted to 4% particle content in 50 mM NaPO₄, 10% sucrose,        0.05% NaN₃, pH 7.4. The deposition flow was 2.5 μL/cm and the        rate was 20 mm/sec.

The deposits were dried for 1 hour at 30° C., and the sheets were cut to0.5 cm wide strips (Matrix 1201 Membrane Cutter, Kinematics Automation).

Coupling of anti-hIgE antibodies to detection particles: Antibodies tohIgE which had been cleaved with pepsin to fab′2 fragments were coupledto fluorescent polystyrene particles having aldehyde groups on theirsurface (Molecular Probes C-17177 TransFluoSpheres, aldehyde-sulphatemicrospheres, 0.1 μm, 633/720, 2% solids). 23 mg of antibody were thencoupled to 66 mg of particles in 50 mM NaPO₄ buffer, pH 6.5, overnightat room temperature, whereupon 205 μL of NaCNBH₄ (5 M) were added toreduce the coupling for 3 hours at room temperature. Centrifugation wasperformed at 20,800×g (50 min in Eppendorf 5417R, 14,000 rpm), anddeactivation in glutamic acid 0.05 M and aspartic acid 0.05 M indeionized water, pH 6.5, was then carried out overnight with stirring atroom temperature. After centrifugation at 20,800×g for 50 min, blockingwas performed with 0.2% BSA in 50 mM NaPO₄, pH 7.4, with 0.05% NaN₃, andincubation took place at +4° C. Centrifugation was then performed againat 20,800×g for 50 min followed by two washes with blocking buffer whichwas then also used for storage. The particle concentration wasdetermined in a fluorimeter (Perkin-Elmer LS50B) with a standard curveprepared with the original particle. The coupled protein during thecoupling was determined by having radioactive anti-hIgE present duringthe coupling.

Biotinylation of hIgE: Biotinylation of hIgE was performed according tothe conditions recommended by the supplier (Pierce). hIgE was desaltedby gel filtration with PD-10 (Amersham Pharmacia Biotech AB) in 0.15 MKPO₄, 0.15 M NaCl, pH 7.8. To 0.95 mL (0.59 mg) hIgE were added 0.010 mLbiotin-LC-Sulfo-NHS (3.59 mM, Pierce). Incubation then took place atroom temperature for 1 hour, whereupon the coupling reaction wasquenched by the addition of 40 μL of 2 M glycine. The mixture was thenapplied to a PD-10 gel filtration column equilibrated with 50 mM NaPO₄,0.15 M NaCl, pH 7.4. Yields and final concentration were calculated fromthe radioactivity as I-125-labelled hIgE was included in the coupling.The concentration of hIgE was analyzed by immunochromatography andUniCAP tIgE (Pharmacia & Upjohn Diagnostics AB, Sweden).

Deposition of biotinylated IgE on application filter: To applicationfilters 5×5 mm (Whatman F075-14) were dispensed 0.006 mL of biotinylatedIgE (1.6 ng) diluted in 50 mM NaPO₄, 0.15 M NaCl, 6% BSA, 5% lactose, 5%dextran T5000, pH 7.4. The filters were dried at 30° C. for 1 hour.

Test procedure: Strips were mounted to a surface inclined about 16° fromthe bench plane. Sucking membranes (3.5 cm wide, Schleicher & Schuell,GB004) were placed 0.5 cm into the upper end of the membrane. To obtainconstant pressure, metal weights were placed on the sucking membranes.Samples and reagents were then pipetted successively as described below.Each sample and reagent volume was sucked into the membrane before thefollowing volume was pipetted.

1) 30 μL of 50 mM NaPO₄, 0.15 M NaCl, pH 7.4.

2) A filter with predeposited biotinylated IgE was placed at the bottomof the strip.

3) 30 μL of patient serum were pipetted to each filter.

4) 20 μL of test buffer (0.1 Na—PO₄, 0.15 M NaCl, 10% sucrose, 3% BSA,0.05% bovine gammaglobulin, 0.05% NaN₃, pH 7.4) were added to thefilter.

5) The application filter was removed.

6) 20 μL of detection conjugate (75 μg/ml) diluted in test buffer.

7) 2×30 μL of test buffer.

8) The fluorescence of the detection zone was measured as a responsearea (Vmm) with a scanning red laser fluorometer (635 nm).

Three positive t3-sera were selected and analysed in triplicate withthree different conjugate batches. Signal areas obtained withnitrocellulose coated with different IgE particle concentrations(described in Example 1 above) run with conjugate 2 were used as astored calibration curve.

PIC correction meant that the signal for the reaction zone for t3 wasmultiplied by PICexp/PICobs before reading against the storedcalibration curve (master curve). PICexp was defined as the average ofthe PIC signals obtained in the run with conjugate 2.

Results Conc (KU/L) Average of triplicate Between Serum Conjugate 1Conjugate 2 Conjugate 3 CV (%) Reading against Master curve foruncorrected signal 1 0.75 3.0 1.4 68 2 5.7 29.1 18.7 66 3 2.5 10.8 6.862 Reading against master curve for PIC-corrected signal 1 2.5 3.5 1.929 2 14.2 19.5 20.7 19 3 3.6 5.3 6.1 26

Between CV (%) is calculated as the variation of the three averagesobtained for the different conjugates. The results show that the idea ofa predeposited calibrator substance in the application zone isfunctional and that the use thereof as a PIC additionally gives areduced between-assay-variation.

1. A lateral flow method for the determination of an analyte in a sampleinvolving utilizing biospecific affinity reactions, and comprising thefollowing steps: i. forming a complex in a lateral flow matrix, thecomplex comprising: Reactant I - - - Analyte′ - - - Reactant*, where a.Reactant* and Reactant I exhibit biospecific affinity to the analyte, b.Reactant* is analytically detectable, c. Analyte′ is the analyte or ananalyte-related reactant, and subsequently ii. determining thedetectable signal constituting a sample value from Reactant* in thecomplex, and iii. determining the amount of analyte in the sample bycomparing the sample value with one or more calibrator values, each ofwhich corresponds to a standard amount of analyte, wherein A) beforedetermination of the calibrator value, either (i) calibrator, or (ii) abinder for the calibrator has been bound to a matrix, and when a binderfor the calibrator has been bound to the matrix, calibrator is added orcalibrator predeposited in the matrix is released for binding with thebinder, and the matrix is insoluble in the liquid medium in whichbinding of Reactant* to the calibrator occurs, B) the calibrator and theanalyte exhibit biospecific affinity to Reactant* by equivalent bindingsites, C) two or more calibrator zones CZ comprising calibrator orbinder for the calibrator are located in a single process flow streamwith Reactant I in a detection zone (DZ), and D) all of the detectionzones DZ are downstream of all of the calibrator zones CZ in the lateralflow matrix.
 2. The method according to claim 1, wherein calibrator hasbeen bound to the matrix before the determination of calibrator value.3. The method according to claim 1, wherein a binder for the calibratorhas been bound to the matrix before the determination of calibratorvalue.
 4. The method according to claim 1, wherein the binder for thecalibrator is one member of a specific binding pair, and the othermember of the specific binding pair is coupled or conjugated to thecalibrator.
 5. The method according to claim 1, wherein a. (i) eachcalibrator zone comprises calibrator in an amount corresponding to astandard amount of analyte, or (ii) each calibrator zone containscalibrator binder, the amount of calibrator binder and the amount ofcalibrator corresponding to a standard amount of analyte, and b.Reactant* is bound to the calibrator by transporting Reactant* throughthe calibrator zones.
 6. The method according to claim 1, wherein alonga single matrix is the flow matrix, and wherein along a single processflow stream, there are a. two or more calibrator zones (CZ), each ofwhich exhibits a matrix calibrator or a matrix calibrator binder, b. oneor more detection zones (DZ), in which a Capturer is firmly anchored andis either Reactant I or a biospecific affinity reactant, which directlyor indirectly binds Reactant I biospecifically, c. an application zonefor Reactant*, A_(R*)Z, which is located upstream of said CZ and DZ andto which Reactant* is optionally predeposited, and d. an applicationzone for sample (A_(S)Z) which is located i. upstream of or coincidingwith a detection zone, ii. downstream or upstream of or coinciding withA_(R*)Z(A_(S)Z/A_(R*)Z), or iii. upstream of, downstream of orcoinciding with a calibrator zone, wherein Reactant* is added to A_(R*)Zif Reactant* is not predeposited, or buffer is added to A_(R*)Z ifReactant* is predeposited, and sample is added to A_(S)Z, optionallypremixed with Reactant* if A_(S)Z and A_(R*)Z coincide, such thatanalyte and Reactant* reach DZ at the same time, or such that analytereaches DZ before Reactant*.
 7. The method according to claim 6, whereinthe calibrator zones CZ comprise a calibrator binder, and calibrator ispredeposited upstream of the calibrator zones.
 8. The method accordingto claim 6, wherein the process flow stream comprises two of saidcalibrator zones, and the level of analyte in the sample is obtained by:a. comparing a calibrator value from one of the calibrator zones locatedin the process flow stream including the detection zone, with one ormore separately obtained calibrator values to determine a deviationbetween the measured calibrator value and the separately obtainedcalibrator values, and b. adjusting the sample value from the detectionzone by the deviation, and subsequently obtaining the level of analytein the sample by comparing the adjusted sample value with one or more ofthe separately obtained calibrator values.
 9. The method according toclaim 6, wherein a. A_(S)Z is (i) common to A_(R*)Z, forming a commonzone A_(S)Z/A_(R*)Z or (ii) is located upstream of A_(R*)Z, and b. foralternative (i) sample is premixed with Reactant* before it is added tothe common zone A_(S)Z/A_(R*)Z, or sample is added to the common zoneA_(S)Z/A_(R*)Z containing predeposited Reactant*, or for alternative(ii), sample is added to A_(S)Z, which is located upstream of A_(R*)Zwhich in turn comprises predeposited Reactant*.
 10. The method accordingto claim 5, wherein Reactant* has particles as an analyticallydetectable group, and/or calibrator or calibrator binder is/are anchoredto the matrix by particles.
 11. The method according to claim 1, whereinthe analyte is an antibody directed to Reactant I or to Reactant*, anda. Reactant* is an antibody directed to the analyte and Reactant I is anantigen or hapten, when the analyte is an antibody directed to ReactantI, or b. Reactant* is an antigen or a hapten and Reactant I is anantibody directed to the analyte, when the analyte is an antibodydirected to Reactant*.
 12. The method according to claim 1, wherein theanalyte is an antigen, and Reactant* and Reactant I are antibodiesdirected to the analyte.
 13. The method according to claim 1, whereinthe method is performed as a part of diagnosing allergy or autoimmunedisease.
 14. A device for transforming measured signal values of acomplexed, analytically detectable reactant (Reactant*) to real amountsof analyte in a sample, in connection with performing an analysis methodwhich utilizes biospecific affinity reactions for the determination ofthe amount of analyte in a sample, wherein the device comprises: a flowmatrix in which there is an area of process flow for the transport ofReactant*, and wherein there are in said area i. two or more calibratorzones (CZ) comprising a calibrator, or binder for the calibrator, whichis firmly anchored to the matrix, the amounts of calibrator orcalibrator binder, respectively, being different for at least twocalibrator zones and the calibrator exhibiting binding sites to whichReactant* binds, when Reactant* is transported through a calibratorzone, ii. an application zone for Reactant* (A_(R*)Z) upstream of saidcalibrator zones, and iii. one or more detection zones (DZ), all of thedetection zones being downstream of all of the calibrator zones.
 15. Thedevice according to claim 14, wherein a calibrator binder is firmlyanchored in the matrix and the device comprises calibrator predepositedupstream of the calibrator zones.
 16. The device according to claim 14,wherein the device comprises Reactant* predeposited in A_(R*)Z.
 17. Thedevice according to claim 14, wherein the process flow comprises adetection zone (DZ) which is located downstream of A_(R*)Z and comprisesa firmly anchored Capturer to which Reactant* can bind in the DZ, and azone of application of sample (A_(S)Z) which is located upstream of orcoincides with said DZ.
 18. The device according to claim 17, whereinA_(R*)Z is located upstream of or downstream of or coincides withA_(S)Z.
 19. The device according to claim 17, wherein the firmlyanchored reactant (Capturer) has biospecific affinity to the analyte orto an analyte-related reactant.
 20. The device according to claim 17,wherein the firmly anchored reactant (Capturer) has biospecific affinityto a second reactant which in turn has biospecific affinity to theanalyte or to an analyte-related reactant.
 21. The device according toclaim 17, wherein A_(S)Z is located upstream of all calibrator zones.22. A test kit, comprising a device according to claim
 14. 23. The kitaccording to claim 22, wherein the kit comprises Reactant*.
 24. The kitaccording to claim 22, wherein the kit comprises calibrator when saiddevice has the calibrator binder bound to the matrix.
 25. The deviceaccording to claim 14, wherein Reactant* has biospecific affinity toanalyte or an analyte-related reactant and to the calibrator.
 26. Themethod according to claim 1, wherein Reactant* comprises a fluorophoregroup or a chromogenic group.
 27. The method according to claim 1,wherein Reactant* comprises metal particles or nonmetal particles. 28.The method according to claim 1, wherein Reactant* comprises gold solparticles.
 29. The device according to claim 14, wherein Reactant*comprises a fluorophore group or a chromogenic group.
 30. The deviceaccording to claim 14, wherein Reactant* comprises metal particles ornonmetal particles.
 31. The device according to claim 14, whereinReactant* comprises gold sol particles.